(a) Field of the Invention
The invention relates to molecular markers for the diagnostic of human diseases such as Crohn's disease (CD).
(b) Description of Prior Art
Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) that can develop anywhere along the gastrointestinal tract. Its etiology is unknown; however, CD is believed to be due to a combination of factors involving diet, genetic background, immunologic responses, and the environment.
In tissues affected by IBD, namely CD and ulcerative colitis (UC), several genes have been shown to be differentially expressed. For example: (i) the expression of mRNA for interleukin-1 (IL-1) and IL-1ra differs in colonic biopsy specimens between IBD patients and inflammatory controls from patients with acute colitis (Isaacs KL et al., Gastroenterology, 1992; 103:1587-95); and, (ii) tumor necrosis factor a (TNF-.alpha.), IL-1B and IL-6 have all been shown to be overexpressed in the inflamed areas of CD specimens as compared to both normal areas and other IBD controls (Reimund J-M et al., Gut 1996; 39:684-89). These genes are all related to the immunological response leading to the inflammation. However, neither the presence of these mRNAs, nor that of their respective proteins, appears useful as specific markers to unequivocally distinguish CD affected tissues from other intestinal diseases.
An important need for the modern medical diagnosis is the development of a clinical molecular marker that positively discriminates a pathology from others. Within this context, the primary aim has been to identify differences in the genetic expression patterns of healthy and diseased tissues. To date, RNA fingerprinting (e.g. differential display and RNA arbitrarily primed PCR such as RAP-PCR) using reverse transcriptase coupled to the polymerase chain reaction (RT-PCR) appears to be the most promising approach for the identification of such molecular markers (Liang P et al., Science, 1992; 257:967-71; McClelland M et al., Trends in Genetic, 1995; 11:242-46; Welsh J et al., DNA and RNA fingerprinting using arbitrarily primed PCR. In: Sninski JJ et al., ed. PCR Protocols, 2nd edition, San Diego, Academic Press, 1994). RNA fingerprinting methods are semi-quantitative and can be used to scan RNA populations for differentially regulated genes based on their relative abundance. Both of these methods have been successful on many occasions, primarily when using RNA isolated from either bacterial or cell cultures that are relatively homogeneous populations. However, several groups working on the characterization of the gene expression associated with clinically important pathologies have been unable to develop any molecular markers using current RNA fingerprint methods. Two causes of these failures are the high heterogeneity of the RNA samples obtained when working with tissues, and the bias introduced by using a minimum number of specimens (e.g. only one diseased and one healthy sample).
It would be highly desirable to be provided with an arbitrarily primed PCR-based procedure designed to identify genetic expression differences between diseased and healthy human tissues using heterogeneous RNA samples.
Also it would be highly desirable to be provided with a marker for the diagnostic of Crohn's disease (CD).